There are few licensed and efficacious broad-spectrum antivirals currently available. Examples include ribavirin, which functions via nebulous effects on both host and virus proteins, and alpha-interferon, which produces unwanted side effects and remains impractically expensive for widespread use (Tam et al., Antivir Chem Chemother, 12:261-72 (2001); Bekisz et al., Growth Factors, 22:243-51 (2004); de Veer et al., J Leukoc Biol, 69:912-20 (2001); Sen, Annu Rev Microbiol, 55:255-81 (2001); Hong and Cameron, Prog Drug Res, 59:41-69 (2002)). The prevailing paradigm in antiviral research emphasizes a “one bug-one drug” strategy; however, the rapid rise in the number of emerging viral pathogens brings into stark contrast the limited resources available to develop therapeutics on a single-pathogen basis (see e.g., Burroughs et al., “The Emergence of Zoonotic Diseases: Understanding the Impact on Animal and Human Health,” Workshop Summary from Board on Global Health, Institute of Medicine, National Academy Press, Washington, D.C. (2002)). The expense and difficulty of developing antiviral drugs tailored to specific pathogens underscores the need to develop broad spectrum antiviral drugs against targets that are common among large classes of viruses.
Viruses can be categorized as either lipid-enveloped or non-enveloped (naked). Enveloped viruses replicate within the host-cell, recruit viral proteins to the host membrane, and then bud from and utilize the host membrane, essentially, as a vehicle to transport the viral genome to new cellular targets. Although the lipid membrane of enveloped viruses derives from the host cell, it differs from host cellular membranes in several biochemical and biophysical properties, such as biogenic reparative capacity, fluidity, lipid composition, and curvature. For example, the membranes of budding viral particles are highly curved relative to the membranes of much larger host cells. As a result, the fusion of enveloped viral particles with new host cells requires that the high curvature viral membranes undergo elastic stresses and subsequent negative curvature needed to promote fusion between the juxtaposed outer lipid monolayers of the viral particles and host cell membranes (Chemomordik et al., J Cell Biol, 175:201-7 (2006); McMahon and Gallop, Nature, 438:590-6 (2005); Chemomordik and Kozlov, Annu Rev Biochem, 72:175-207 (2003)).
The central role of virus-host cell fusion in the infectivity of enveloped viruses has motivated the development of small molecule antiviral therapeutics that insert, intercalate, or otherwise bind to viral membranes and disturb the membrane dynamics required for successful virus-host cell fusion (e.g., Chemomordik et al., J Cell Biol, 175:201-7 (2006); Martin and Ruysschaert, Biochim Biophys Acta, 1240:95-100 (1995); Langosch et al., J Biol Chem, 276:32016-21 (2001); Langosch et al., Cell Mol Life Sci, 64:850-64 (2007)). For example, the phospholipid analog lysophosphotidylcholine (LPC) is designed to prevent the entry of certain enveloped viruses, such as influenza, HIV-1 (Class I fusion) and TBEV (Class II fusion), into host cell by stabilizing the positive spontaneous curvature of viral membranes and thereby preventing conformational changes needed for viral-host cell fusion (Chemomordik et al., J Cell Biol, 175:201-7 (2006); Chemomordik and Kozlov, Annu Rev Biochem, 72:175-207 (2003); Martin and Ruysschaert, Biochim Biophys Acta, 1240:95-100 (1995); Razinkov et al., J Gen Physiol, 112:409-22 (1998); Shangguan et al., Biochemistry, 35:4956-65 (1996); Gunther-Ausborn et al., J Biol Chem, 270:29279-85 (1995)). However, LPC's viability as a drug candidate is questionable since it exerts its effect in a highly reversible manner, requires high molar concentrations (10% or higher total lipid content), and can be effectively recycled and metabolized by cells.
n-docosanol, a 22-carbon saturated alcohol, is also designed to inhibit host cell entry of a variety of enveloped viruses (Katz et al., Proc Natl Acad Sci USA, 88:10825-9 (1991)). However, n-docosanol appears to inhibit virus-cell fusion by perturbing the properties of the target cell rather than the virus (Katz et al., Ann N Y Acad Sci, 724:472-88 (1994); Marcelletti et al., AIDS Res Hum Retroviruses, 12:71-4 (1996); Pope et al., Antiviral Res, 40:85-94 (1998)), as optimal inhibition is observed when cells, but not virus, are pre-incubated for several hours with n-docosanol. In addition, poor solubility and a millimolar IC50 has limited n-docosanol to use as a 10% v/v topical microbicide (Abreva™) for the treatment of cold sores (Katz et al., Ann N Y Acad Sci, 724:472-88 (1994); Marcelletti et al., AIDS Res Hum Retroviruses, 12:71-4 (1996); Pope et al., Antiviral Res, 40:85-94 (1998)).
Recently, amphipathic peptides derived from the NS5A protein of Hepatitis C have been identified as having antiviral activity related to their ability to disrupt the membrane integrity of enveloped viruses (Bobardt et al., Proc Natl Acad Sci USA, 105:5525-30 (2008); Cheng et al., Proc Natl Acad Sci USA, 105:3088-93 (2008)). Although the NS5A-derived peptides were not active against all enveloped viruses, their broad range of activity validates viral membranes as a therapeutic target for broad spectrum antiviral therapeutics.
Thus, there is a need in the art for broad-spectrum antiviral drugs capable of disrupting viral lipid membranes and thereby preventing viral infections.
Toxoplasma gondii is an obligate intracellular parasite in the phylum Apicomplexa that causes severe central nervous system disorders of immunocompromised (AIDS/transplant/lymphoma) individuals, birth defects in congenitally infected neonates, and ocular disease in immunocompetent persons. In addition to being an important pathogen in its own right, Toxoplasma serves as a model system for studying apicomplexan parasites which cause a number of diseases of medical and veterinary importance worldwide. There are over 5000 different species of apicomplexans, but the most notable of these is Plasmodium falciparum, the causative agent of malaria which kills 1-2 million people each year. Other Apicomplexans which cause disease in humans include the opportunistic pathogens Cryptosporidia spp. and Isospora belli. Important veterinary pathogens include Neospora caninum (a pathogen of dogs/cattle), Theileria spp (cattle), and Eimeria spp (poultry).
Apicomplexan parasites share a common mechanism of gliding motility that drives invasion into the host cell, a process that is essential for survival. The parasites secrete an array of molecular adhesins from a specialized secretory organelle named the micronemes onto the surface of the parasite which mediate motility and attachment to the host cell. The cytoplasmic tails of these adhesins interact with a parasite actin:myosin motor which is anchored in a membrane system underlying the plasma membrane known as the inner membrane complex. Action on the actin:myosin motor provides the driving force for motility, and also for the parasites to actively penetrate their respective host cells. In Toxoplasma and Plasmodium, the micronemal protein AMA1 also has been shown to organize the moving junction RON proteins which are secreted from another organelle called the rhoptries and this complex of proteins is critical for subsequent invasion. Current antiparasite therapies target biosynthetic pathways in the parasite and the bacterial-like apicoplast and are prone to mutations leading to resistance.
Thus, there is a need in the art for broad-spectrum antiparasitic drugs that are less susceptible to resistance through mutation.